The long term goal is to understand in molecular terms how a neurotropic herpes virus invades and spreads in the mammalian nervous system. Most alpha herpesviruses (e.g., herpes simplex virus HSV; varicella-zoster virus, VZV; and pseudorabies virus, PRV, display a striking neurotropism infecting both the peripheral and central nervous system, and do so in every animal species that can be infected. In PRV, two genes (gE, gI) are required for anterograde, but not retrograde spread in the nervous system of living animals. A new gene (Us9) was recently discovered that also is required for anterograde spread of PRV. All three gene products also influence virulence. Research in this proposal will continue a focused genetic and biochemical attack to determine the functions of the gE, gI and Us9 gene products in invasion, spread and pathogenesis in the nervous system. Four specific aims are proposed to test a general model of directional spread of virus in the nervous system: In aim 1, the localization and trafficking of gE, gI and Us9 proteins in cultured neurons and infected nervous system tissue will be determined. In aim 2 alanine substitution mutations in the gE ectodomain will be analyzed to define sites of interaction with putative cellular receptors. In addition, the role of the gI cytoplasmic tail on gE function will be determined. In aim 3, a genetic and biochemical analysis of Us9 will be initiated to determine its role in anterograde trans-neuronal spread. Finally, in aim 4: the role of gD, a viral ligand essential for virion infection, will be analyzed to determine if gD is involved in gE/gI/Us9-dependent and -independent spread of virus in defined neuronal circuits. The knowledge to be learned from completion of this work is important because the direction taken by the virus in a neuron after primary infection or reactivation from latency, as well the extent of spread in a neuronal circuit can be the difference between a minor peripheral infection or a lethal viral encephalitis.